The use of tobacco products is the leading cause of preventable deaths in the world, leading to approximately 5 million deaths per year. The link between tobacco use and disease is not surprising given, 1) the addictive property of nicotine that promotes continued use of tobacco despite the severe health consequences and 2) the presence of more than 55 carcinogens in tobacco. In order to reduce and eventually eliminate the profound impact of tobacco use on society, it is necessary to elucidate the molecular details of nicotine dependence with the goal of identifying novel therapeutic approaches. Nicotine exerts its effects by binding to and activating neuronal nicotinic acetylcholine receptors (nAChRs), cognate receptors of the endogenous ligand acetylcholine (ACh). Activation of the receptors in the dopaminergic (DAergic) mesocorticolimbic reward pathway is thought to underlie the initiation of addiction whereas signaling through nAChRs in the habenulo-interpeduncular pathway that feeds into the reward pathway is thought to play a key role in eliciting nicotine withdrawal symptoms. Functional neuronal nAChRs are made up of homomeric or heteromeric combinations of ? and ? subunits, which include ? 2 - ? 10 (encoded by the genes designated CHRNA2-10) and ? 2 - ? 4 (CHRNB2-4). Five subunits assemble and insert into the plasma membrane forming a cation-selective channel. A critical point is that the precise subunit composition determines the functional properties of the receptor. Our laboratory has extensively studied the transcriptional mechanisms underlying expression of the CHRNA3, A5 and B4 genes. Interestingly, these three nAChR genes are tightly clustered in the genome (in the order CHRNA5/A3/B4 relative to the centromere) and their products assemble to form a major subtype of nicotinic receptors expressed in the nervous system. Importantly, a recent series of linkage analyses, genome-wide association studies and candidate gene analyses has identified multiple single nucleotide polymorphisms across the CHRNA5/A3/B4 locus that are associated with nicotine-related behaviors such as age of initiation, cigarettes smoked per day, lung cancer and particularly germane to this application, nicotine dependence. In this R21 application, we propose to study post-transcriptional regulation of the CHRNA5/A3/B4 locus with a specific focus on microRNAs (miRNAs). miRNAs are emerging as key modulators of post-transcriptional gene regulation, particularly within the nervous system. However, little is known regarding the role of miRNAs in nAChR expression. To date, there is a single report of a miRNA regulating nAChR expression. This study showed that miR-1 regulates nAChR expression in body muscle of C. elegans. Nothing has been reported regarding miRNA regulation of nAChR in mammals. The goal of this proposal is to use a multidisciplinary approach to test the hypothesis that expression of the CHRNA5/A3/B4 nAChR genes are regulated by miRNAs and further, that such miRNAs are themselves regulated by nicotine. We have used a combination of miRNA library screening and in silico analysis to identify several miRNAs that regulate CHRNA5/A3/B4 expression in HEK293T cells. We have also identified potential binding sites for several of these miRNAs in the 3'- untranslated regions of the receptor subunit genes. Finally, preliminary data indicate that expression of at least one of the candidate miRNAs is up-regulated by chronic nicotine treatment. These data are the basis of the Specific Aims of this proposal, which are, 1) To test the hypothesis that nAChR ? 3, ? 5 and/or ? 4 subunit expression is regulated by miRNAs and 2) To test the hypothesis that nicotine regulates expression of miRNAs identified in Aim 1, particularly within the DAergic mesocorticolimbic and habenulo-interpeduncular circuits. If our hypotheses are correct, we will have established miRNAs as key regulators of the nicotine-dependence susceptibility locus, CHRNA5/A3/B4, and set the stage for a more in-depth analysis of the roles miRNAs may play in nicotine-associated behaviors.